DESCRIPTION

CD86 (also known as B7-2) is a costimulatory molecule belonging to the immunoglobulin (Ig) superfamily. CD86 is primarily expressed on antigen-presenting cells (APCs), including B cells, dendritic cells, and macrophages. CD86 has strong structural similarity with another B7 family molecule, CD80 (B7-1). CD86 and CD80 are the ligands for two proteins at the cell surface of T cells, CD28 antigen and cytotoxic T-lymphocyte antigen 4 (CTLA-4). Binding of CD86 or CD80 with CD28 antigen is a costimulatory signal for T cell activation, proliferation, and cytokine production. Binding of CD86 or CD80 with CTLA-4 negatively regulates T cell activation and diminishes the immune response. CD86 and CD80 bind to CTLA-4 with higher affinity than CD28. Defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	1,462.8	59.4	4.1
2	8	354.6	11.1	3.1
3	8	83.8	3.9	4.6

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,542.7	130.7	8.5
2	16	368.4	22.8	6.2
3	16	85.8	6.0	7.0

RECOVERY

The recovery of human CD86 spiked to three different levels throughout the range of the assay in human serum was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:4	103	93-117
	1:8	111	104-116

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human serum
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	115
	Range (%)	113-117
1:8	Average% of Expected	103
	Range (%)	102-104
1:16	Average% of Expected	93
	Range (%)	87-99

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CD86 produced at Sunlong Medical™.